In vitro Activity of Garlic (Allium sativum€¦ · known edible plants such as onion, chives,...

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*Corresponding author: Email: [email protected];

European Journal of Medicinal Plants4(10): 1240-1250, 2014

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In vitro Activity of Garlic (Allium sativum) onSome Pathogenic Fungi

Elham Abdelbasit Suleiman1* and Wafa Ballal Abdallah2

1Veterinary Research Institute, Mycology Department, Amarat, Khartoum, Sudan.2Faculty of Science, University of Khartoum, Sudan.

Authors’ contributions

Author EAS Designed the study, wrote the protocol, and the first draft of the manuscript.Author WBA managed the literature search and performed the statistical analysis.

Both authors read and approved the final manuscript

Received 16th March 2014Accepted 25th June 2014Published 15th July 2014

ABSTRACT

Aim: This study was conducted to investigate the in vitro antifungal activity of garlic(Allium sativum) on some pathogenic fungi.Study Design: This is a comparative evaluation report on garlic as an antifungal agent.Place and Duration: Department of mycology, Veterinary Research institute, betweenJune-October 2013.Methodology: Samples of garlic were obtained from a local market. It was thoroughly,cleaned, peeled and pulverized. Aqueous and organic extracts of garlic were obtained bymaceration and Soxhlet extractor apparatus. The methanol and petroleum ether extractswere tested against Candida albicans, Aspergillus, Curvularia and some Dermatophytespecies using cup diffusion and agar incorporated methods. Diameter of Inhibition zonesof growth were measured in millimeter (mm) and expressed as Mean ±SD.Results: The obtained results revealed that aqueous and petroleum ether extractspossess the stronger activity and a broader fungicidal spectrum against tested fungicompared to methanol extract. The study also showed that the dry coarsely- powderedgarlic was found to be more potent to Candida albicans than the commercial Nystatin.Conclusion: The study demonstrated the potent activity of garlic against tested fungiwhich encourages its use as a suitable alternative drug for controlling fungal infectionsbecause it has far less risk of side-effects than most known antifungal drugs and it canbe used indefinitely in quite large amounts. Therefore, adding garlic to food (raw) or

Original Research Article

European Journal of Medicinal Plants, 4(10): 1240-1250, 2014

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crushing and swallowing raw cloves which are cheaper is recommended as a powerfulanti-fungal agent. Further purification and formulation of the garlic would give a trueantifungal activity comparable to standard antibiotics.

Keywords: Garlic; antifungal; inhibition zone; Candida; dermatophytes; Aspergillus.

1. INTRODUCTION

Medicinal herbs are the use of natural herbs and plant's extracts for the treatment orprevention of diseases, disorders and promotion of good health [1,2]. It has a long traditionuse outside of conventional medicine [3]. In the Sudan, the use of herbal treatment has beenwell known over the country since a long time ago. Due to the mutilating surgical andprolonged medical treatment by drugs, which are very expensive and have side effects,people started looking back on native treatment [4].

Garlic (Allium sativum) is one of the herbs that have been used worldwide. It is a species inthe onion genus, Allium [5]. With the history of human use of over 7,000 years, garlic isnative to central Asia and has long been a staple in the Mediterranean region, as well as afrequent seasoning in Asia, Africa, and Europe [6]. It is a member of the familyAmaryllidaceae , Subfamily Allioideae which contains over 6,000 species including well-known edible plants such as onion, chives, leek, and shallot [5]. In Sudan, the northernregions are considered the most productive areas for garlic. The ancient Egyptians, Greeks,Chinese, Indians and Romans all advocated the therapeutic value of garlic in the treatmentof ailments. It has been used for both culinary and medicinal purposes [6]. The biologicalactivity of garlic was determined. The bactericidal effect of fresh garlic juice was tested byLouis Pasteur onto growing bacterial colonies. It was shown that fresh garlic juice inhibitedthe growth of most gram-positive and negative bacteria. The action was comparable in vitrowith penicillin, streptomycin, chloramphenicol, tetracycline and erythromycin [6]. Previousstudies further demonstrated that ethanol and aqueous extract exhibited growth inhibition ofmost gram positive and negative as well as multidrug resistant bacteria [7,8].

The chemistry of the Allium species has been dominated by many sulfur containingcompounds that give them a characteristic flavour and exhibit potent anti-fungal properties[9]. Among the most studied are allicin, alliinase and allin (S-allyl cysteine sulfoxide), theprecursor of allicin which upon crushing of garlic bulb, it is hydrolyzed by the enzymeallinase to its active form allicin [10]. It is responsible for most of the biological property ofgarlic. One gram of allicin (S-methyl-l-cysteine sulfoxide has been equated to 15 IU ofpenicillin in its antibiotic activity. However, a variety of non sulfur compounds, worksynergistically to provide various health benefits [11]. Freshly pressed juice of garlic has astrong antifungal effect on the major pathogenic moulds, yeast and dermatophytes [12].The action of garlic on yeast and fungi is perhaps even more dramatic. Some studiesrevealed garlic as effective as fluconazole [8] at inhibiting Candida albicans. Odorlesscapsules, liquid extract and tablets were available for treating intestinal yeast infection. Freshgarlic was significantly more potent against Candida albicans. The essential oil, water, andethanol extracts, and the juice also, inhibited the in vitro growth of Candida and Aspergillusspecies [12]. Moreover, Ajoene is also known to have effective broad antifungal propertieshelpful in preventing yeast infection (Candida albicans) and treating athlete's foot (Tineapedis) [13]. Garlic changes its characteristics because of the complexity of its intrinsicchemistry, and processing procedures [11]. Thus, standardization marker compounds areimportant for ensuring consistent effects.


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Garlic oil and recently available stabilized allicin, revealed a considerable activity againstvarious phytopathogenic fungi. In a field study with peanut, garlic oil was found to beprotective against Gibberella zeae, (a fungal parasite of maize). It was inhibited by garlic oil(8,000-10,000ppm) more effectively than by pesticides. Moreover, larvae of Trogodermagranarium Everts, another parasite on maize kernels, was controlled by 1-2% garlic oil. Thepresent study aims to evaluate antifungal activity of garlic against pathogenic fungi.

2. MATERIALS AND METHODS

2.1 Test Microorganisms

Eight fungal strains were used. Aspergillus niger (ATCC9763), Candida albicans(ATCC7596) were obtained from National Council for Research, Khartoum, Sudan. Clinicalisolates: Aspergillus flavus, Microsborum canis, Curvularia lunata, Microsporum audouinii,Tricophyton mentagrophytes, Tricophyton soudenense were obtained from the stock of theMycology Department, Veterinary Research Institute, Khartoum, Soba, Sudan. They weremaintained on Sabouraud’s Dextrose Agar (SDA) media.

2.2 Sample Preparation

Fresh bulbs of garlic were purchased from a local market in Khartoum North, Sudan. Thecloves were separated, peeled and washed to obtain the edible portion. 1000 grams werecrushed using mortar and pestle. They were kept under shade for two days to dry. Theywere pulverized with a blender to a fine powder and kept for further analysis.

2.3 Extraction of Garlic Powder

Extraction was carried out according to the method described by Harborne [14]. One kg ofdried plant sample was successively extracted with (2.5L) of petroleum ether and 80%methanol using Soxhlet extractor. The extraction was carried out for about 4hr and 8hr,respectively. Solvents were evaporated under reduced pressure and temperature below theirmelting point using Rotary evaporator. Finally, the extracts were allowed to dry in petridishes until complete dryness. The obtained extracts were weighed and kept in a refrigeratorfor further study. The percentage yield was calculated using the formula below:

% Yield=Weight of extract / Weight of plant sample * 100

2.4 Preparation of the Aqueous Extract

Hundred grams of the garlic sample were soaked in 500 ml of hot distilled water, and left tillcooled down with continuous stirring at room temperature. The extract was filtered usingwhatman n filter paper no.1 to give crude aqueous extract of 200mg/ml. This was collectedin a sterile vial and kept in deep-freeze. The Freeze extract was then dried using freeze-drier till powdered extract obtained and kept at 4ºC until used [14].

2.5 Preparation of Samples for Antimicrobial Analysis

Four samples were used for this study: crude methanol extract, petroleum ether, aqueousand dry fraction. One gram of each sample was weighed and dissolved in 10ml of thesolvent used for the extraction to give 100mg ml-1. This was serially diluted until a


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concentration of 6.25 mg ml-1 of the content was obtained. Different concentrations of thedry-powdered garlic (50mg, 100mg, and 200 mg) were prepared and incorporated into theSabouraud’s Dextrose Agar (SDA) media after sterilization. Control groups were runsimultaneously in order to check their growth inhibitory effects.

2.6 Agar Dilution Method (MIC)

2.6.1 MIC=Minimal inhibition concentration

One gram of ether extract was added to 10ml of the solvent. Serial dilutions of extract weremade by transferring 5ml from tube1 to tube2 containing 5ml of solvent. The two-fold dilutionwas continued up to tube3. Each dilution was tested on A. flavus, C. albicans and somedermatophytes. The concentration of garlic at which no growth of the organism wasobserved was considered as the minimum inhibitory concentration (MIC) of garlic for thatorganism. The results were expressed in terms of growth of organisms (+) or inhibition ofgrowth (-) after addition of the extract [15].

2.7 Preparation of Fungal Inoculums

Inoculum for Candida albicans, was prepared from overnight growth (18 h) to 48 h oldcultures on SDA. 2ml amounts of sterile distilled water were inoculated with a loop full of theisolate. For filamentous fungi sterile distilled water with 0.05% Tween 80 was added to thesurface growth and the spores and hyphae were scraped off with a sterile wire loop. Theconcentration of the suspension was adjusted to about 1x106 cells/ml. Suspensions werestored in the refrigerator until used.

2.8 In vitro Testing of Antimicrobial Activity of the Extracts

2.8.1 The cup-plate agar diffusion method

The cup-plate agar diffusion method Perez [16] was adopted with some minor modificationsto assess the antifungal activity of the prepared extracts. One gram of each extract wasweighed and dissolved in 5ml of the solvent used for the extraction to give 200mg /ml 0.1ml of 100 mg/ml the standardized fungal stock suspension was thoroughly mixed with 20 mlmolten sterile Sabouraud dextrose agar and maintained at 25ºC. The agar plates were left toset and in each of these plates 4 cups (10mm in diameter) were cut using a sterile cork borer(No. 4) and agar discs were removed. Cups were filled with 0.2ml of each extract usingautomatic microlitre pipette, and allowed to diffuse at room temperature for two hours. Theplates were then incubated in the upright position at 37ºC for 2-3 days. Activity of eachextract was tested in triplicate. The diameters of zones of inhibition were measured inmillimeter using a transparent well calibrated ruler, averaged and the mean values weretabulated.

2.8.2 Agar incorporation technique

The anti-fungal susceptibility of the extracts and the standard drugs ketoconazole andNystatin were determined using agar incorporation technique in Sabouraud’s Agar Mediumsupplemented with 0.05mg/ml chloramphenicol and 0.5mg/ml cycloheximide. Each platewas inoculated with the test organism. The control group was inoculated with the sameorganism. All plates were incubated at 250C for three to seven days except for


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dermatophytes which were incubated for up to three weeks at the same temperature [17].Different concentrations of 50, 100 and 200mg/ml dry coarsely powdered garlic were also,tested using incorporated technique. Parallel experiments were run in the same way usingsolvent vehicle (petroleum ether and 80% methanol).

2.9 Statistical Analysis

The mean values were expressed as the Mean± Standard Deviation (SD) and wereanalyzed using one-way ANOVA using the program SPSS version19.0 for WindowsDifferences were considered significant at P<0. 05.

3. RESULTS

The percentage yield of garlic extract obtained is shown in Table (1).

Table 1. Percentage yield of garlic obtained after extraction.

Solvent Weight (gram) Yield %Methanol (80%) 40.62 4.00Petroleum ether 1.08 0.10

In the present study the antifungal activity was investigated. Previous studies demonstratedthat garlic has a broad range of antifungal activity. Our result further supported this finding.Both aqueous and petroleum ether extracts displayed antifungal activity using cup diffusiontest. Clear zones of inhibition were observed. Curvularia lunata was found to be moresensitive (45mm) than other tested fungi (Fig. 1). Zones of inhibition on petroleum etherextract against tested fungi are shown in (Table (2). While the methanol extract showed noeffect.

Table 2. Antimicrobial activity of garlic extracts against C. albicans and molds at aconcentration of 10mg/ mL.

Organism Methanol Petroleum ether Aqueous extractDiameter of inhibition zone (mm)

Candida albicans 0.0 23±0.06 16±1.41Aspergillus niger 0.0 16±1.09 14±1.63Aspergillus flavus 0.0 38±1.26 23±0.82Curvularia lunata 0.0 45±1.34 45±1.15

Inhibition zone diameters are expressed as Mean ± (SD).

Agar incorporated method displayed antifungal activity of the extracts demonstrated by –vegrowth on SDA treated plates. Petroleum ether and aqueous extracts of garlic were found toinhibit the growth of the standard organism of Candida albicans and Aspergillus niger andthe clinical isolates of A. flavus, Curvularia lunata, Microsporum audouinii, Trichophytonsoudanense and Trichophyton mentagrophytes at a concentation of 10mg/ml (Table 3; Figs2&3). The dry coarsely- powdered crude garlic was found to show the strongest antifungalactivity than the organic and aqueous extracts against tested fungi. Different concentrationsof the dry crude garlic, 2.5mg, 5mg, and 10mg/ml were found to affect the growth of Candidaalbicans, Microsporum audouinii, Microsporum canis and Tricophyton soudanense. The


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minimum concentration inhibited the growth was found to be 2.5mg/ml (Table 4). The vehicle(petroleum ether) revealed resistance to tested fungi (Fig. 4).

Fig. 1. Zone of inhibition (mm) of Curvularia lunata on petroleum ether extract.Ca: Candida albicans, M: methanol extract of garlic, P: petroleum ether extract of garlic

Table 3. Antimicrobial activity of garlic extracts by agar incorporated method at aconcentration of 10 mg/ml of the extract.

Organism Methanol Petroleum ether AqueousCandida albicans + _ _Aspergillus niger + _ _Aspergillus flavus + _ _Curvularia lunata + _ _Microsporum audouinii + _ _Tricophyton mentagrophytes + _ _

+ indicates growth of fungi (-ve response) ,_ indicates no growth (+ve response)


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Fig. 2. Growth of Trichophyton soudanense on SDA control plate (right) and inhibitionof growth on petroleum ether extract (left) plate at a concentration of 10mg/ml.

Tre= treated plate, Co= control plate

Fig. 3. Growth of Microsporum canis on SDA control plate( right) and inhibition ofgrowth on petroleum ether extract (left) plate at a concentration of 10mg/ml


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Table 4. Antifungal activity of dry crude powdered garlic on some pathogenic fungi

Organism 2.5mg/ml 5mg/ml 10mg/mlC. albicans + + +M. audouinii + + +M. canis + + +T. soudanense + + +

+ indicates sensitivity of garlic.

Fig. 4. Growth of T. mentagrophytes (left), growth of M. audouinii (right) on petroleumether treated plates at a concentration of 10mg/ml

Significant antifungal effect was expressed as MIC of petroleum ether extract against testedfungi is shown in Table 5. MIC value of 2.5mg/ml was found to show the strongest activity ofpetroleum ether extract against test microorganisms. It inhibited the growth of Aspergillusand dermatophyte species tested except C. albicans where MIC value of 10mg/ml was foundto inhibit its growth.

Table 5. Minimum inhibitory concentration (MIC) of petroleum ether extract of garlic

Organism Petroleum ether extract mg/ml10 5 2.5

Candida albicans _ + +Aspergillus niger _ _ _Aspergillus flavus _ _ _Curvularia lunata _ _ _M. audouinii _ _ _T. mentagrophytes _ _ _

- indicates +ve response (sensitivity) + indicates –ve response (resistance)

4. DISCUSSION

Garlic (Allium sativum) is a spice with global recognition. In the present study, it has beenshown to inhibit the growth of fungi when in vitro tested. The antimicrobial activity of garlic is


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believed to be due to the effect of allicin, the main ingredient in garlic, generated by thephosphopyridoxal enzyme allinase [18] and ajoene [19]. Previous studies reported that allicinis a pure, bioactive and the most powerful medicinal compound isolated from garlic [19]. Ithas strong antimicrobial and antifungal activities. Thus, inhibition of fungi observed in thisstudy may be related to allicin or ajoene which curbs the performance of some enzymes thatare important to fungi [19].

In the present study, the methanol extract of garlic has no effect on fungi tested. This mightbe due to insolubility of the phytoconstituents ingredients in methanol. Whereas, petroleumand aqueous extracts displayed greater antifungal activity against C. albicans and testedmolds. Similar results were obtained by previous coworkers [8,18] where inhibition of C.albicans using fresh crushed homogenate garlic was shown. Our study demonstrated highpotency of petroleum ether extract against Curvularia lunata. Singh and his colleaguesdisclosed similar result of inhibition of spore germination of Curvularia lunata due to ajeonefraction [20] as germination of hyphae was used as indicator in suspect testing of broth andagar method [14].

Inhibition of growth of dermatophytes observed in this study was similar to previous findings.Several studies by Dikasso et al. [12] and yoshda et al. [19] had previously demonstratedantifungal potency of garlic where inhibition of Trichophyton and Microsporum species usingfresh garlic juice was shown due to stronger activity of ajoene. Mason and his colleaguessuccessfully treated athelets foot using garlic relating that to ajoene fraction of garlic [9].Moreover, all extracts of garlic in higher concentrations showed that the antifungal effectsincreased with increased concentrations [8]. However, the crude coarsely powdered garlichad greater antifungal activity compared to other extracts. This is due to the complexchemistry of Allium plants as variations in processing, yield quite different preparations thus,highly reactive thiosulfinates, such as allicin which disappear during processing and arequickly transformed to other types of organo-sulfur compounds. So, efficacy and safety arecontingent upon processing methods [11]. Hence, some of the antimicrobial function mightbe lost. This finding supports the suspicion of Hassan and his collaborators [21] whobelieved that the whole herbs contain many ingredients, and they may work together toproduce a beneficial effect. This fact supports our finding where dry-powdered garlic wasfound to have stronger antifungal effect. This might be due to damage caused by garlic tothe outer surface of the fungal cells together with alterations in the fat content of the cell. It isalso probable that garlic may reduce the adhesion of fungal cells to epithelial membrane.Moreover, the organo sulphur compound present in garlic (allicin) is thought to inhibit fungalgrowth via interaction with sulphur containing enzymes [19]. The main antimicrobial effect ofallicin is due to its chemical reaction with thiol groups of various enzymes such as alcoholdehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essentialmetabolism of cysteine proteinase activity [18].

Comparing antifungal effect of crude dry-powdered garlic with Nystatin and Clorox, it wasfound that the dry-powdered garlic was most potent against C. albicans. It was shown thatthe antifungal activity of garlic exceeds that of nystatin and Clorox. Previous study disclosedsame result [5]. This might be due to the assumption that the very best form of garlic is theraw and crushed, preferably certified the organic extract. When raw garlic is crushed it startsa chemical process which creates allicin which exhibits potent anti-fungal properties [9].

Moreover, comparative studies on the effects of garlic juice and the pharmaceuticalpreparations, nystatin, against Candida albicans, have shown that the antimycotic activity of


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garlic exceeds that of all the drugs investigated [5]. This finding supports our result. Thus,use of fresh garlic or its extract is highly recommended as a potent anti-fungal agent.

5. CONCLUSION

Petroleum ether, methanol and water extracts of garlic were investigated for theirantimicrobial activity. Petroleum ether, and aqueous extracts were shown to have greatantifungal activity against dermatophytes, C. albicans and other mould tested. The crude drygarlic was shown to have the strongest antifungal activity than aqueous and petroleum etherextracts. The antifungal potential of dry garlic against C. albicans exceeds that of thecommercial Nystatin. The study indicated that garlic has strong antifungal activity. Furtherresearch on purification and formulation may be needed to understand the mechanismsthrough which this effect is exerted.

CONSENT

Not applicable.

ETHICAL APPROVAL

Not applicable.

ACKNOLEDGEMENS

The authors are indebted to Sudan Academy of Science (SAS) for permission to carry thisstudy. Technical assistance of Mycology Department Staff is highly appreciated.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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_________________________________________________________________________© 2014 Suleiman and Abdallah; This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

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In vitro Activity of Garlic (Allium sativum€¦ · known edible plants such as onion, chives,...

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